Effect of recombinant protein expression on baculovirus budded virus structure and infectivity

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Baculovirus expression vectors (BEVS) have become one of the most versatile and successful systems for the production of recombinant proteins. Frequently, they succeed in producing difficult targets where other systems have failed. Increasingly, they are employed to produce recombinant protein subunit vaccines, e.g. influenza, Covid-19 and several for animal diseases. They are also very useful for producing large, complex transmembrane proteins for drug discovery.The natural replication cycle of baculoviruses in insect cells involves early, late and very late phases of gene expression. Notably, the late phase sees the production of infectious virus particles that bud from the plasma membrane and are essential for virus dissemination in cell culture. Recombinant proteins are largely produced in the very late phase, however, some protein synthesis initiates in the late phase and if the target is cytotoxic this can have an impact on budded virus (BV) yield. This may reduce the infectious titre of virus by up to 10-fold. While this is not usually a problem in small scale tests to monitor recombinant proteins, it can have serious consequences in subsequent scale up work where large cultures of insect cells are infected with BEVS using high particle:cell ratios. A further issue is the production of BV stocks with high titres that may not be stable upon storage. Rather than maintaining a high titre for months, this period reduces to a few weeks of storage in a cold room. The cause(s) of the virus low titre and instability are unknown, apart from the link with recombinant protein production. We hypothesise that the structural integrity of BV is affected by the recombinant protein. However, BV structural data is very scarce due to limited access to state-of-the-art facilities, leading to a lack of in-depth understanding of these expression vectors and impacting on further BEVS development. With world-leading structural-characterisation facilities at STFC RAL and ISIS we can examine and investigate the structural integrity of recombinant BV with compromised infectious titre or stability on storage. The successful completion of this structural study will allow us to formulate intelligent design strategies for recombinant protein production that mitigate their effect on BV structure and infectivity. Thiswill allow projects that currently fail at the feasibilioty stage to progress to scale up and protein production for many different academic and commercial uses.